NRG1 (neuregulin 1) is a member of the EGF family, and particularly soluble NRG1 binds to ErbB3 or ErbB4 and functions as a ligand of these receptors. Additionally, ErbB3 and ErbB4 are members of the EGF (epidermal growth factor) receptor family. It is also known that when soluble NRG1 binds to these receptors, the structures of the receptors change, forming homodimers or heterodimers. Further, such dimerization leads to phosphorylation of intracellular domains of these receptors. The phosphorylated intracellular domains further bind to signaling proteins, activating a variety of signal transduction events. Then, such NRG1-involved signal transduction regulates a series of biological reactions such as cell proliferation, cell differentiation, apoptosis, and cell migration, adhesion, and infiltration. More concretely, it has been revealed that NRG1 is involved in the growth and development of nervous systems and heart, as well as various cancers such as breast cancer, ovarian cancer, colorectal cancer, stomach cancer, lung cancer, thyroid cancer, gliomas, medulloblastoma, and melanoma.
Furthermore, soluble NRG1 is derived from a transmembrane precursor, like other factors belonging to the EGF family. To be more specific, it is believed that after produced as a transmembrane precursor (pro-NRG1), soluble NRG1 is transported to the cell surface (plasma membrane), and a cleavage enzyme such as metalloprotease cleaves (sheds) and releases a membrane neighboring portion of an extracellular domain of the pro-NRG1. There is a report on the pro-NRG1 cleavage mechanism that the cleavage and release of pro-NRG1 by a protease at the cell surface are activated by a factor in a serum or PMA (phorbol-12-myristate-13-acetate): an activation factor for protein kinase C (PKC) (NPL 1). Further, two such proteases have been reported so far: TACE (tumor necrosis factor-α (TNFα) converting enzyme, ADAM17) and ADAM19 (meltrin β) (NPLs 2 and 3). It has been shown that PKC, Erk1/2, p38, and the like are involved in the mechanism of activating these proteases (NPL 4).
Meanwhile, nine NRG1 protein isoforms have been known. Among these, isoform 1 (NRG1-β1), isoform 2 (NRG1-α), isoform 3 (NRG1-β2), and the like are the aforementioned transmembrane precursors. Further, these isoforms are roughly classified into two types: α type (isoform 2) and β type (isoforms 1 and 3).
As described above, the NRG1-involved signal transduction regulates a variety of biological reactions. Hence, analyzing or controlling the signal transduction and the cleavage that would otherwise trigger the signaling for each isoform greatly contributes to the elucidation of a life phenomenon and eventually to the development of the medical field. In this regard, it is known generally that antibodies can be effective tools in specifically analyzing protein isoforms and controlling the functions thereof.
However, as shown in FIG. 1, α type and β type isoforms of the NRG1 protein substantially match for a portion from the N-terminus to an amino acid residue at position 212. The differences are just in a 10 amino-acid residue long C-terminal part of an EGF domain, and only approximately 20 amino acids of a juxtamembrane domain (which is a region located on the C-terminal side of the EGF domain and on the N-terminal side of the transmembrane sequence, and where the cleavage takes place). Further, the α type and β type isoforms match for the interval of six cysteine residues included in a C-terminal portion of the EGF domain. Thus, it is quite difficult to prepare an antibody capable of identifying such slight differences, so that an antibody capable of specifically recognizing these isoforms and also suppressing signal transduction in which these isoforms are involved has not been developed under current situations.
On the other hand, since it has been revealed that NRG1 is involved in various life phenomena as described above, antibodies against NRG1 have been developed, although the antibodies do not specifically bind to isoforms (NPLs 5 to 8).
For example, NPL 5 discloses that an anti-NRG1 antibody inhibits the mitogenic response of Schwann cells. In NPL 6, an anti-NRG1 antibody was able to suppress the cell division of schwannoma cells (benign tumor cells), suggesting a possibility to treat schwannoma using an anti-NRG1 antibody. Moreover, NPL 7 discloses that adding a culture supernatant of SK-Hep1, which is hepatocellular carcinoma (HCC)-derived cells, enhanced phosphorylation of ErbB3 in HepG2, which is also HCC-derived cells, but the phosphorylation was not enhanced by a culture supernatant pre-treated with an anti-NRG1 antibody. Further, NPL 8 discloses an antibody (3G11) which exhibits a neutralizing activity against the growth stimulation by NRG1 isoform 6 (SMDF). Nevertheless, it has also been revealed that, even with such an antibody exhibiting a neutralizing activity, no inhibitory effect was observed against MCF7 and T47D, which are cancer cells (see the description of the fifth line from the bottom in the left column at page 248 of this literature to the line 13 in the left column at page 248). As such, under current situations, no antibody against NRG1 has been developed which has a sufficient activity in the treatments of various diseases in which NRG1 is presumably involved, particularly in the cancer treatments.